1. Field of the Invention
Affinity chromatography has recently become valuable in research involving biological materials. It is particularly valuable for the purification of various biologically active molecules such as small ligands, proteins, nucleic acids and enzymes, etc. In affinity chromatography, a substrate is immobilized on the support during the chromatography. By utilizing a column of the immobilized substrate, materials having affinity or binding specificity for the compounds bonded to the stationary phase can be separated from other materials in a mixture.
Heretofore, complex polysaccharide materials such as agarose beads, etc., were employed in affinity chromatography. A suitable ligand is bonded to the polysaccharide matrix through one of a variety of methods. By passing a liquid mixture containing molecules which have a binding affinity for the ligand on the column, the materials which have affinity for the ligand will be preferentially bound to the insoluble and stationary substrate. The bound material may then be eluted by passing a solution through the column which reduces the binding affinity of the material for the ligand.
The insoluble or stationary phase material should preferably interact very weakly with the materials to be separated so as to minimize non-specific adsorption. The insoluble material should ideally comprise uniform, spherical particles exhibiting good flow and mechanical properties and which retain their insoluble characteristics after conjugation with a particular ligand. The substrate should be mechanically and chemically stable under all conditions of affinity chromatography methods. In addition, the insoluble phase should form a loose, porous network which permits uniform flow of the mixture to be fractionated through the entire structure.
The most common material utilized today as the stationary substrate for affinity chromatography is agarose or other polysaccharide materials. These materials, however, do not possess ideal flow characteristics and tend to pack under pressure into a solid mass making uniform flow of the mixture to be fractionated therethrough difficult. In addition, the polysaccharide materials employed today are not sufficiently stable under all conditions employed in liquid chromatography of biological materials thereby shortening their useful life span.
One particularly troublesome disadvantage associated with polysaccharide supports derives from the fact that it is usually necessary to employ cyanogen halides to bond the ligand thereto. The utilization of cyanogen halides, however, results in the formation of isourea groups in the substrate thereby giving rise to an anion-exchange capacity therein. The presence of these isourea groups renders the substrate non-useful for the purification by affinity chromatography of acidic enzymes (i.e., those with an isoelectric point of 4-6). Cyanogen halide treated polysaccharide supports are also incompatible with organic solvents such as acetone, chloroform and benzene and are subject to microbioal attack.
A further disadvantage associated with polysaccharide supports is that they are unsuitable for use in high pressure liquid chromatography applications. In these methods, the material to be fractionated as well as the elution media, etc., are forced through the column under high pressure thereby greatly shortening the overall separation time. The polysaccharide supports are totally unsuitable for use in these applications since they tend to pack together into an impermeable mass under pressure. The use of high pressure techniques would be especially advantageous in the purification of biologically active molecules such as enzymes, etc. due to the instability of most biologically active molecules. Most gravitational flow affinity chromatography methods require long periods of time, i.e., 1-3 days, to complete, thereby rendering them unsuitable for the treatment of highly unstable enzymes, etc. Solid supports, such as glass beads, would appear to be ideally suited for absorbent media in affinity chromatography methods, particularly those operated under high pressures. Their great rigidity and strength characteristics would render them ideal for packing chromatographic columns.
Workers active in the field, however, have indicated that glasses are unsuitable. Lowe et al ("Affinity Chromatography," John Wiley, London, 17, 1974) state that glasses absorb strongly basic and some neutral proteins and that this presents a serious hazard to their widespread use in selective functional purifications. Zaborsky ("Separation and Purification Methods," Vol. 3, 1 (1974)) states that glass supports are "soluble" to some extent at high pH. In addition, no satisfactory method has been proposed for coating glass beads with ligand-containing materials suitable for utilization in affinity chromatography.
It is an object of the present invention to provide intermediates, copolymers containing suitable ligands and insoluble, stationary phase substrates bonded to said copolymers for utilization in affinity chromatography methods.
These and other more widespread objects will become apparent from the following description of the invention.
2. Description of the Prior Art
In addition to the above-noted publications, the following reference describes various aspects of the art of affinity chromatography:
Cuatrecasas et al, "Advances in Enzymology," 36, 29 (1972)
U.S. Pat. No. 3,715,343 to Slagel et al disclose various polymers of vinyl aminimides.